PROJECT SUMMARY Chronic obstructive pulmonary disease (COPD), which encompasses airway obstruction and/or emphysema, is a growing cause of morbidity and mortality in HIV+ individuals in the current era. Pathogenesis of HIV- associated COPD is poorly understood, but shifts in the lung microbiome may play a role. The lung interacts with the host immune system and receives constant exposure to the environment and to microorganisms aspirated from the oral cavity. We do not yet know how these exposures might impact the lung and how microorganisms in the lung might differ in HIV and in COPD. The current study will leverage data and biospecimens from the Lung HIV Microbiome Project (LHMP), the first multicenter study of the lung microbiota. This project will add to the LHMP framework by undertaking a microbiome-based (microbial communities, their gene content, and environmental interactions) examination of the lung while simultaneously developing or enhancing the use of high throughput ?omics-based technologies. First, we will examine how host recognition of microorganisms differs between HIV-infected and uninfected individuals and individuals with and without COPD. Using LHMP bronchoalveolar lavage (BAL) specimens, we will identify key bacteria and fungi that drive host response using a novel magnetic activated cell sorting (MACS) assay to target and separate immunoglobulin (Ig)-bound bacteria and fungi. We will then use sequencing techniques to compare taxonomy of Ig-bound bacteria between individuals with and without HIV and with and without COPD. Selected chemokines and cytokines (small signaling molecules) produced by the human host will also be measured to provide a snapshot of host immune system status. We will then characterize the biological functions of the microbiome more broadly using whole community RNA to measure metabolic activity of not only bacteria, but also potential contributions from fungi, archaea and bacteriophage. A series of comparative bioinformatic and statistical approaches will be used to characterize and compare community structure and function between individuals with and without HIV and COPD. Development of these methods will reveal not only shifts in taxonomic structure in relation to host immune recognition, but through sequencing RNA, also identify biological function. Understanding collective microbial community function will allow us to better address the question of how does the metabolism of the community that resides in the lung of individuals with HIV and COPD differ from individuals without these conditions, and in what ways may their metabolic activity contribute to changes in lung function. Collectively, this study will examine lung microbial communities in individuals with differing HIV and COPD status, forming the foundation for larger-scale mechanistic studies while developing methodology directly applicable to other microbiome-based lung studies of health and disease.